The coagulation of blood involves a cascading series of reactions leading to the formation of fibrin. The coagulation cascade consists of two overlapping pathways, both of which are required for hemostasis. The intrinsic pathway comprises protein factors present in circulating blood, while the extrinsic pathway requires tissue factor, which is expressed on the cell surface of a variety of tissues in response to vascular injury. Davie et al., 1991, Biochemistry 30:10363. Agents that interfere with the coagulation cascade, such as heparin and coumarin derivatives, have well-known therapeutic uses in the prophylaxis of venous thrombosis. Goodman and Gilman, eds., 1980, The Pharmacological Basis of Therapeutics, MacMillan Publishing Co., Inc., New York.
Tissue factor (TF) has been investigated as a target for anticoagulant therapy. TF is a membrane glycoprotein that functions as a receptor for factor VII and VIIa and thereby initiates the extrinsic pathway of the coagulation cascade in response to vascular injury. In addition to its role in the maintenance of hemostasis by initiation of blood clotting, TF has been implicated in pathogenic conditions. Specifically, the synthesis and cell surface expression of TF has been implicated in vascular disease (Wilcox et al., 1989, Proc. Natl. Acad. Sci. 86:2839) and gram-negative septic shock (Warr et al., 1990, Blood 75:1481).
Ruf et al. (1991, Thrombosis and Haemostasis 66:529) characterized the anticoagulant potential of murine monoclonal antibodies against human TF. The inhibition of TF function by most of the monoclonal antibodies that were assessed was dependent upon the dissociation of the TF/VIIa complex that is rapidly formed when TF contacts plasma. Such antibodies were thus relatively slow inhibitors of TF in plasma. One monoclonal antibody, TF8-5G9, was capable of inhibiting the TF/VIIa complex without dissociation of the complex, thus providing an immediate anticoagulant effect in plasma. Ruf et al. suggest that mechanisms that inactivate the TF/VIIa complex, rather than prevent its formation, may provide strategies for interruption of coagulation in vivo.
The therapeutic use of monoclonal antibodies against TF is limited in that currently available monoclonals are of rodent origin. The use of rodent antibodies in human therapy presents numerous problems, the most significant of which is immunogenicity. Repeated doses of rodent monoclonal antibodies have been found to elicit an anti-immunoglobulin response termed human anti-mouse antibody (HAMA), which can result in immune complex disease and/or neutralization of the therapeutic antibody. See, e.g., Jaffers et al. (1986) Transplantation 41:572. While the use of human monoclonal antibodies would address this limitation, it has proven difficult to generate large amounts of human monoclonal antibodies by conventional hybridoma technology.
Recombinant technology has been used in an effort to construct “humanized” antibodies that maintain the high binding affinity of rodent monoclonal antibodies but exhibit reduced immunogenicity in humans. Chimeric antibodies have been produced in which the variable (V) region of a mouse antibody is combined with the constant (C) region of a human antibody in an effort to maintain the specificity and affinity of the rodent antibody but reduce the amount of protein that is non-human and thus immunogenic. While the immune response to chimeric antibodies is generally reduced relative to the corresponding rodent antibody, the immune response cannot be completely eliminated, because the mouse V region is capable of eliciting an immune response. Lobuglio et al. (1989) Proc. Natl. Acad. Sci. 86:4220; Jaffers et al. (1986) Transplantation 41:572.
In a recent approach to reducing immunogenicity of rodent antibodies, only the rodent complementarity determining regions (CDRs), rather than the entire V domain, are transplanted to a human antibody. Such humanized antibodies are known as CDR-grafted antibodies. CDRs are regions of hypervariability in the V regions that are flanked by relatively conserved regions known as framework (FR) regions. Each V domain contains three CDRs flanked by four FRs. The CDRs fold to form the antigen binding site of the antibody, while the FRs support the structural conformations of the V domains. Thus by transplanting the rodent CDRs to a human antibody, the antigen binding domain can theoretically also be transferred. Owens et al. (1994) J. Immunol. Methods 168:149 and Winter et al. (1993) Immunology Today 14:243 review the development of CDR-grafted antibodies.
Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833 constructed a humanized antibody against the relatively simple hapten nitrophenacetyl (NP). The CDR-grafted antibody contained mouse CDRs and human FRs, and exhibited NP binding activity similar to the native mouse antibody. However, the construction of CDR-grafted antibodies recognizing more complex antigens has resulted in antibodies having binding activity significantly lower than the native rodent antibodies. In numerous cases it has been demonstrated that the mere introduction of rodent CDRs into a human antibody background is insufficient to maintain full binding activity, perhaps due to distortion of the CDR conformation by the human FR.
For example, Gorman et al. (1991) Proc. Natl. Acad. Sci. 88:4181 compared two humanized antibodies against human CD4 and observed considerably different avidies depending upon the particular human framework region of the humanized antibody. Co et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869 required a refined computer model of the murine antibody of interest in order to identify critical amino acids to be considered in the design of a humanized antibody. Kettleborough et al. (1991) Protein Engineering 4:773 report the influence of particular FR residues of a CDR-grafted antibody on antigen binding, and propose that the residues may directly interact with antigen, or may alter the conformation of the CDR loops. Similarly, Singer et al. (1993) J. Immunol. 150:2844 report that optimal humanization of an anti-CD18 murine monoclonal antibody is dependent upon the ability of the selected FR to support the CDR in the appropriate antigen binding conformation. Accordingly, recreation of the antigen-binding site requires consideration of the potential intrachain interactions between the FR and CDR, and manipulation of amino acid residues of the FR that maintain contacts with the loops formed by the CDRs. While general theoretical guidelines have been proposed for the design of humanized antibodies (see, e.g., Owens et al.), in all cases the procedure must be tailored and optimized for the particular rodent antibody of interest.
There is a need in the art for humanized antibodies with reduced immunogenicity and comparable binding affinity relative to the parent rodent antibody for various therapeutic applications. In particular, there is a need for a humanized antibody against human tissue factor having anticoagulant activity and useful in the treatment and prevention of thrombotic disease.